Akhilesh PaperBioactive component, cantharidin from Mylabris cichorii and its antitumor activity...

8/2/2019 Akhilesh PaperBioactive component, cantharidin from Mylabris cichorii and its antitumor activity against Ehrlich ascit

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ORIGINAL RESEARCH

Bioactive component, cantharidin from Mylabris cichorii

and its antitumor activity against Ehrlich ascites carcinomaAkalesh Kumar Verma & Surya Bali Prasad

Received: 4 August 2011 / Accepted: 1 December 2011# Springer Science+Business Media B.V. 2012

Abstract The anticancer activity of the extract of

blister beetle, Mylabris cichorii has been documented

earlier by us. In the present study, the active principle

of M. cichorii was isolated and its anticancer efficacy

was evaluated against murine Ehrlich ascites carcinoma

(EAC). The isolated bioactive compound was charac-

terized to be cantharidin which showed potent antitumor

activity and inhibited the proliferation of Ehrlich ascites

carcinoma, both in vivo and in vitro. Cantharidin-treated

EAC-bearing mice showed about 82% increase in life-

span at the dose of 0.5 mg/kg/day. In vitro cytotoxicity

assay with the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphe-

nyltetrazolium bromide test revealed about 50% cell

death at the concentration of 25.8 g/ml. The fluores-

cence and transmission electron microscopy revealed

that EAC cells treated with cantharidin depicted typical

apoptotic morphology with chromatin condensation,

nuclear fragmentation into discrete masses, and plasma

membrane blebbing which deduce towards the death of

these cells. Histological examination of the kidney of

cantharidin-treated mice showed glomerular and tubular

congestion with abnormal Bowmans capsule, thus, in-

dicating a renal toxicity in the host. Cantharidin-induced

renal damage in the host was also manifested by the

decreased lactate dehydrogenase isozymes and its pos-

sible release from the cells.

Keywords Apoptosis . Anticancer activity .

Cantharidin . Ehrlich ascites carcinoma.Mylabris

cichorii . Toxicity

Abbreviations

CC Column chromatography

EAC Ehrlich ascites carcinoma

ILS Increase in lifespan

IR Infrared

MTT {3-(4,5 dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide}

NMR Nuclear magnetic resonance

PBS Phosphate-buffered saline

TEM Transmission electron microscope

TLC Thin layer chromatography

Introduction

The wide ranges of plants and animals have attracted

the human kind for their use as traditional, folk med-

icine throughout the world (Roja and Heble 1994;

Gupta et al. 2004). Ingredients sourced from plants

and animals are not only used in traditional medicines,

but are also increasingly valued as raw materials in the

Cell Biol Toxicol

DOI 10.1007/s10565-011-9206-6

A. K. Verma: S. B. Prasad (*)

Cell and Tumor Biology Laboratory,

Department of Zoology, North-Eastern Hill University,

Shillong 793022, India

e-mail: [email protected]


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preparation of modern medicines and herbal prepara-

tions (Shoeb 2006; Alves and Alves 2011). The treat-

ment of human ailments with remedies made from

animals and their products is called zootherapy (Wang

1989; Alves and Rosa 2005). The animal-derived rem-

edies as an integral part of folk medicine may constitute

an important alternative among many other known ther-apies practiced worldwide. Various animals and its parts

have been used for treating different diseases such as

asthma, rheumatism, wounds, thrombosis, bronchitis,

epilepsy, cancer, renal failure, etc. (Alves and Rosa

2007). In India, nearly 1520% of the Ayurvedic med-

icine is based on animal-derived substances (Mahawar

and Jaroli 2008).

There are many reports on the use of animals or

animal-derived products in the treatment against

cancer-suspected disease. Anticancer peptide of mo-

lecular mass 6,280 Da was isolated from Buthus mar-tensii Karsch that prevented proliferation of the mouse

S-180 fibrosarcoma cells and murine Ehrlich ascites

carcinoma (EAC) cells (Kapoor 2010). It has been

reported that charybdotoxin, 37 amino acid neurotoxin

from the venom of the scorpion Leiurus quinquestria-

tus hebraeus, induces depolarization in human breast

cancer cells, arrests the cells in the early G1, late G1,

and S phase and accumulated cells in the S phase

(Cavallucci et al. 2010). The skin extract of common

Indian toad Bufo melanostictus, schneider exhibits

significant antineoplastic activity on EAC cells andhuman leukemic cell lines U937 and k562 (Upadhyay

and Ahmad 2010).

Beetles are one of the insects of medical impor-

tance, mainly due to the presence of broad spectrum of

chemical substances within their hemolymph. The

blister beetle, Mylabris cichorii has been used by the

traditional healers of some parts of Assam, India for

the treatment of cancer suspected diseases. The anti-

tumor potential of this beetle extract against murine

ascites Daltons lymphoma has been reported earlier

by us (Prasad et al. 2010b).Most of the reported work on the importance of

beetles deals with Chinese blister beetles Mylabris

phalerata (Pall.) and Spanish fly, Lytta vesicatoria.

Chinese blister beetle, M. phalerata is 1530 mm long

and 510 mm wide. Each elytron has a large orange

yellow spot at its base where it joins the thorax and

also two wide transverse orangeyellow bands. Both

bands and the black background have stiff black hairs

(Singh 2001). The Spanish fly, L. vesicatoria is actually

not a fly, but a member of the blister beetle family and it

is an emerald green beetle, 1522 mm long and 58 mm

wide (Moed et al. 2001).

M. cichorii is found in parts of India as well as

China. M. cichorii is about 1220 mm long and 3

6 mm wide. The bands of the elytra are pale oechre

yellow and the basal one often joins the middle band along the inner margin of each elytron. Yellow

hairs occur upon the yellow bands and black hairs on the

black background (Singh 2001). These beetles belong to

the Family Meloidae, Order Coleoptera in class Insecta.

Various reports have shown the presence of cantharidin

in the blister beetles (Moed et al. 2001; Bonness et al.

2006; Rauh et al. 2007). In contemporary studies, can-

tharidin has been shown to be active in cervical, tongue,

gingival, mucoepidermoid carcinoma, adenocystic car-

cinoma, neuroblastoma, bone, ovarian, and colon cancer

cell lines among others (Wu et al. 1992; McCluskey etal. 2000; Sakoff et al. 2002)

The present study was carried out on the blister

beetle, M. cichorii which is very commonly found

in our studies areas and very less work has been

done on the beetles of this region. Moreover, the

details of its active compound and its effect on

EAC cells have not been explored. Therefore, in

an attempt to recognize the anticancer active prin-

ciple of M. cichorii, the present study was under-

taken to isolate the bioactive compound from these

beetles and evaluate its effect on the EAC cells invivo and in vitro. The findings from the present

studies demonstrate that the bioactive compound in

the M. cichorii is cantharidin, which exhibits po-

tent anticancer activity and induces apoptosis in

EAC cells.

Materials and methods

Animals and tumor model

Inbred Swiss albino mice were maintained under con-

ventional laboratory conditions (20 2C) with free

access to food (Amrut Laboratory, New Delhi) and

water ad libitum. EAC is being maintained in vivo in

1012-week-old mice by serial intraperitoneal (i.p.)

transplantations of 1106 viable EAC cells (Gothoskar

and Ranadive 1971) per animal (0.25 ml in phosphate-

buffered saline (PBS), pH 7.4). Tumor-transplanted

mice usually survived for 1820 days. The use of

Cell Biol Toxicol


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animals in the present study was as per the ethical

norms and has been cleared by the institutional

ethical committee of North-Eastern Hill University,

Shillong, India.

Collection and identification of M. cichorii

Blister beetle, M. cichorii was collected from different

locations of Karbi Anglong and North Cachar Hills

districts of Assam, India. Species identification was

done in Zoological Survey of India, Kolkata, bearing

identification report no 9/2007 and a voucher speci-

men (no. SBP 101) was deposited in the department of

Zoology, North-Eastern Hill University, Shillong,

India.

Extraction and purification of the active component

The powdered beetle (1 kg) was extracted three

times with 2 l of absolute methanol each time. The

accumulated extract was concentrated under re-

duced pressure using rotary evaporator at 40C.

The dried free-flowing sample (80 g) was sub-

jected to column chromatography, using a 45

4 cm glass column filled with silica gel 60 (mesh

size, 60120) in n-hexane. Prepared methanol ex-

tract sample was added to the free volume at the

head of the column. Total 75 fractions were collect-

ed using n-hexane: ethyl acetate (1:10 and finallywith 100% ethyl acetate) as the eluting solvents and

each fraction was tested for the activity against

tumor model. Based on the similar thin layer chro-

matography (TLC) profile (Rf values) and high

antitumor activity, fractions 1625 were combined

and single active compound (white crystals) was

purified using second-column chromatography over

silica gel eluted with n-hexane: ethyl acetate (1:15 and

1:20). The purity of compound was confirmed by TLC

and proton nuclear magnetic resonance (1H-NMR).

Spectral measurements of the isolated compound

Infrared spectrum was recorded in chloroform on a

Perkin-Elmer system 2000 Fourier-transformed infra-

red (IR) Spectrophotometer calibrated against the

polystyrene absorption at 1,601 cm1. Mass spectrum

was recorded on a gas chromatographymass spectrom-

etry in a Bruker Daltonic Data Analysis 2.0 Spectrom-

eter. 1H-NMR (300 MHz) and 13C NMR (75 MHz)

spectra were recorded using CDCl3 as solvent in a

Bruker Advance DPX-300 NMR machine considering

TMS as an initial standard and chemical shift values

were in delta parts per million ( ppm) values. Spectra

were referenced to tetramethylsilane (1H) or solvent

(13C) signals.

Antitumor activity study

Cantharidin was initially dissolved in dimethyl sulf-

oxide (DMSO) at a concentration of 4 mg/ml and

stored at 4C. Its anticancer activity was determined

following the method described by Ahluwalia et al.

(1984). Tumor cells were transplanted intraperito-

neally in 1112-week-old male mice (30 g) and the

day of transplantation was taken as day 0. The tumor-

transplanted animals were randomly divided into eight

groups with 10 mice in each group. On the sixth day oftumor transplantation, mice were treated with different

doses of cantharidin (0.5, 1, 1.5, and 2 mg/kg body

weight/day; i.p.) and the LD50 was determined based

on these doses. Subsequently sublethal doses of can-

tharidin were selected and diluted with PBS to get the

desired concentration, i.e., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,

0.7 and 0.8 mg/kg body weight/day. The mice in

various groups were treated with different concentra-

tion of cantharidin for five consecutive days starting

from the sixth day of tumor transplantation. The con-

trol group of tumor-bearing mice received the samevolume of cantharidin vehicle (the same volume of

DMSO diluted with PBS) alone. The deaths of ani-

mals, if any in different treatment groups, were

recorded daily. The anticancer efficacy was deter-

mined in percentage of average increase in life span

(%ILS) using the formula: (T/C100)100, where, T

and C are the mean survival days of treated and

control groups of mice, respectively. The dose of

cantharidin, i.e., 0.5 mg/kg body weight showing

highest anticancer activity was selected for further

apoptotic study using transmission electron micro-scope (TEM) and fluorescence microscope.

To have a comparative analysis of the cantharidins

antitumor effect, in other set of experiment, a known

anticancer drug, cisplatin (2 mg/kg body weight/day,

i.p.) was given as the reference drug to the tumor-

bearing mice on the sixth day of transplantation daily

up to tenth day. The cisplatin has been used as a

reference anticancer drug by other workers also (Ajith

and Janardhanan 2003).

Cell Biol Toxicol


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In vitro cytotoxicity assay (MTT)

Cell growth inhibition was determined by {3-(4,5

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide} (MTT) assay. MTT assay is a nonradioactive

colorimetric assay (Campling et al. 1988) to measure

cell cytotoxicity, proliferation, or viability. Briefly1106 cells in 1-ml culture medium were seeded on

24-well plates and the cells were treated with different

concentration (10, 20, 30, 40, and 50 g/ml) of canthar-

idin for 12 h. At the end of the incubation, culture

medium was removed and MTT (5 mg/ml) was added

and the cells were further incubated for 4 h. After

removing the media, DMSO (100l) was added in each

well to solubilize the formazan crystals. The absorbance

was read at a wavelength 595 nm. Cell death was

expressed as percentage over the control. The same trea-

ted cells were also processed for in vitro apoptosis assayusing acridine orange and ethidium bromide (AO/EtBr)

staining method as described below.

Apoptosis study using fluorescence microscopy

Fluorescence-based in vivo apoptosis was determined

by using AO/EtBr staining method as described by

Shylesh et al. (2005). After 24, 48, 72, and 96 h of the

treatment of the EAC-bearing mice with cantharidin,

tumor cells were collected, washed with PBS, and trea-

ted with AO/EtBr (100 g/ml PBS of each dye). Thecells were thoroughly studied under fluorescent micro-

scope (Leica) using a blue filter and photographed.

Viable cells nucleus stain green due to permeability of

only acridine orange whereas, apoptotic cells appear

yellowred due to costaining of both stains.

Transmission electron microscopy

The EAC cells from mice in different groups were col-

lected and processed for transmission electron microsco-

py as described by Prasad et al. (2010a). Briefly, each cellsuspension was mixed rapidly with an equal volume of

2% glutaraldehyde solution in 0.1 M cacodylate buffer

and fixed for 2 h. The cells pellet obtained after centrifu-

gation (1,000gfor 5 min) was resuspended twice in an

excess of 0.1 M cacodylate buffer with a 15-min interval.

Cells were resuspended in 1% osmium tetroxide in 0.1 M

cacodylate buffer and fixed for 30 min and then centri-

fuged at 1,000g for 5 min. Then, 0.1 M cacodylate

buffer was added and this step was repeated twice. The

samples were stored in 2% glutaraldehyde solution at

4C until further processing for embedding, cutting

ultrathin sections, and viewing under transmission

electron microscope JEOL 100CX II.

Kidney histopathology

For the analysis of kidney toxicity, normal mice (25

30 g) were divided into three groups with 10 mice in

each group. Mice in group I, serving as normal con-

trol, received (i.p.) vehicle alone from days 1. Mice

in group II, serving as toxic control or positive control,

received single dose of cisplatin (8 mg/kg of body

weight; i.p.) as described by Prasad et al. (2006). In

group III, serving as treated group, mice were admin-

istered with cantharidin (i.p., 0.5 g/kg of body

weight/day) for 6 days. Mice in different groups were

killed after 14 days of the treatment and kidneys werecollected for histopathological studies as described by

Yang et al. (2006). Slices of the left kidney (from five

animals of each group) were fixed in 10% formalin for

48 h and were embedded in paraffin. Thin sections

(45 m thick) collected on glass slides were depar-

affinized and stained with hematoxylin and eosin

stain. The stained sections were examined under a

light microscope (Leica DFC425 C) and the cellular

features and any deformities were recorded.

Lactate dehydrogenase isozymes profile

To understand further on the cantharidin-induced dam-

age/toxicity on kidney, lactate dehydrogenase (LDH)

isozymes pattern and its intensity pattern was also

determined for the kidney, which were collected and

used for histology. Polyacrylamide slab gel (6%) was

prepared and electrophoresis was performed following

the method of Davis (1964). Tissue homogenate (20%

in PBS, pH 7.4) was prepared and centrifuged at

8,000g for 15 min at 4C and the supernatant was

collected. Equal amount of (40 l, i.e., 25 mg protein)tissue homogenate supernatants were loaded on the

gel. After electrophoretic separation, the gel was pro-

cessed for LDH-specific staining. The gel was dipped

in LDH specific reaction solution (10 mg NAD, 10 mg

MTT, 1 mg PMS, and 2 ml of 60% L-lactate as substrate

in 50 ml millipore water), and incubated at 37C for

15 min and the reaction was stopped by adding tap water

and the gel was fixed and stored in 7% acetic acid. The

net band intensity analysis of all five LDH isoforms

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(LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5) was

carried out using Transilluminator Bioview UXT-20 M-

8E Gel logic 100 imaging system.

Statistical analysis

The results were expressed as meanSD. Statistical

significance was determined by one-way analysis of

variance. The difference among multiple groups was

analyzed by a post hoc test, Bonferroni. Pvalue 0.05

were considered as statistically significant.

Results

Isolation, characterization, and structural elucidation

of active compound

TLC profile [ethyl acetate/chloroform (1:10)] under

UV light showed the presence of total 15 spots; where-

as in visible range, only three spots were visible in

methanol crude extract. The anticancer activity was

shown by the single purified compound having Rfvalue 0.78 [ethyl acetate/chloroform (1:10)]. The

instrumental analysis data of isolated active compound

is shown in Table 1.On the basis of spectroscopic data

mentioned above and comparing with the literatures

(Walter and Cole 1967; Wang et al. 2000), this isolatedcompound sample code Cry 01 was identified as can-

tharidin and showed the purity over 98% (TLC and

1H-NMR). The spectrogram for IR, TLC, and NMR

profiles has been shown in Fig. 1.

Antitumor activity study

The determination of LD50 value from the different doses

of the isolated cantharidin was found to be 1 mg/kg bodyweight in Swiss albino mice (SB Prasad, personal com-

munication). Out of the different sublethal doses of

isolated cantharidin used, the dose of 0.5 mg/kg was

found to be the most effective against EAC. The effect

of cantharidin at this dose on the survival of tumor-

bearing mice is shown in Table 2. Mean survival time

for the control group was about 20 days, which increased

to about 36 and 37 days for the groups treated with

cantharidin (0.5 mg/kg/day) and cisplatin (2 mg/kg/

day), respectively. The increase in the lifespan of

tumor-bearing mice treated with cantharidin and cisplatinwas found to be about 82% and 87%, respectively, as

compared to the control (Table 2).

In vitro cytotoxicity assay (MTT)

The effect of cantharidin on viability of tumor cells

was checked using the MTT assay. The cantharidin

treatment decreased the viability of the EAC cells in a

dose-dependent manner as shown in Fig. 2. Cantharidin

at about 25.8 g/ml decreased the viability of EAC cells

to 50% of the initial level and this was chosen as theIC50. However, in case of cisplatin, it was 32 g/ml.

Longer exposures resulted in additional cytotoxicity to

Table 1 Physical and spectral data for the purified compound (cantharidin) from M. cichorii

Sl no. Parameter data

1 Sample code Cry 01

2 Yield 7% w/w

3 Nature Crystalline

4 Color White

5 Solubility Chloroform, alcohol, ethyl acetate and dimethyl sulfoxide

6 Rfvalue (TLC) 0.78 [Ethyl acetate/chloroform (1:10)]

7 Molecular formula C10H12O4

8 Molecular weight 196.1

9 EIMS (m/e,% 70ev) 96 (100%), 128 (83%), 70 (28%), 109 (11%), 95.1 (19%)

10 IR (Chloroform) cml, 3000 (CH), 17801850 (C0O), 1240 (CO)

11 13C-NMR (CDCL3, 75 MHz) 12.69 (CH3), 23.42 (C-5, C-6), 55.26 (C-3a, C-7a), 84.74 (C-4, C-7), 175.99 (C-1, C-3)

121

H-NMR (CDCL3, 300 MHz) 1.248 (6H, s, CH3), 1.827 (2H, m, H-5, H-6), 4.734 (2H, t, J04.9 Hz, H-4, H-7)

TLC thin layer chromatography, IR infrared, NMR nuclear magnetic resonance

Cell Biol Toxicol


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the cells. Comparison of the doses of cantharidin and

cisplatin and the determination of cytotoxicity by in vitro

MTT assay suggest that cantharidin seems to be more

effective/cytotoxic to EAC cells as compared to the

reference drug cisplatin after 12 h of exposure (Fig. 2).

Apoptosis study using fluorescence microscopy

Acridine orange is a vital dye that stains both live and

dead cells, whereas ethidium bromide will stain only

those cells that have lost their membrane integrity

(Shylesh et al. 2005). Cells stained green represents

viable cells, whereas yellowishred staining represents

apoptotic cells. The control EAC cells were rounded in

shape with deep green fluorescence in blue filter

(Fig. 3a). After 24-h treatment, nuclei constriction

and early apoptotic features were very much prominent

(Fig. 3b) while at 48 h of treatment, reduction in cell

volume, cell shrinkage, and loss of cell membrane in-

tegrity and appearance of membrane blebbing were

observed (Fig. 3c). At 72 h of incubation period, severe

nucleus fragmentation was observed in more than 70%of cells with many late apoptotic cells and few early

apoptotic cells. At 96 h of treatment, changes in cellular

Fig. 1 Spectrogram for

infrared (IR), thin

layer chromatography

(TLC) and nuclear magnetic

resonance (NMR) profile

of the pure isolated

compound, cantharidin.

a Infrared (IR) spectropho-

tometry profile. b13

C- NMRshowing the number of

carbon atoms. c1

H-NMR

showing the number of

proton. d TLC profile

of crude extract showing

many spots (lane 1) and a

single spot for isolated

pure compound (lane 2)

Table 2 Effect of cantharidin treatment on mean survival time

and percentage ILS of Ehrlich ascites carcinoma-bearing mice

Groups Treatments

(mg/kg)

Mean survival

time (days)

% Increase in

life span (ILS)

Control Vehicle 201.3

Cisplatin 2 37.52.5* 87.50

Cantharidin 0.5 36.451.2* 82.25

Values are mean SD, n06. Significance of difference between

control and treated groups was tested by one-way ANOVA

*P0.001, significant with respect to control

Fig. 2 Cytotoxicity of cantharidin against Ehrlich ascites carci-

noma cells determined by MTT assay after 12 h of incubation at

different doses. Control group is treated with vehicle alone

whereas, cisplatin is used as a positive reference drug. Results

are expressed as meanSD. ANOVA, n05, *P0.05 as com-

pared to cisplatin treatment

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morphology, including chromatin condensation, mem-

brane blebbing, fragmented nuclei, large size cytoplas-

mic, and membrane vacuoles were seen with completeloss of membrane integrity (Fig. 3e). Thus, the morpho-

logical features of cantharidin-treated EAC cells showed

the involvement of apoptosis.

The percentage of apoptotic cells in vitro at different

doses of cantharidin and cisplatin for 12 h exposure is

shown in Fig. 4. Cisplatin-treated cells also showed the

apoptotic morphology but the apoptotic cells were com-

paratively lower at different doses as summarized in

Fig. 4. Here, cell deaths were observed but nucleus

fragmentation and membrane blebbing was not visible.

Transmission electron microscopy

Here, TEM study was carried out to corroborate the

observations made by AO/EtBr staining. Ultrastruc-

tural examination of the cantharidin-treated EAC cells

showed typical morphological features of apoptosis

(Fig. 5). The morphological changes observed were re-

duction in cell volume, cell shrinkage, reduction in chro-

matin condensation, and nucleus fragmentation. Control

Fig. 3 AO/EtBr staining of EAC cells. a Control, EAC cells from

the mice treated with vehicle alone is roundedin shape, with green

fluorescence. After the treatment of mice with cantharidin (0.5 mg/

kg body weight) for 24 h, b EAC cells depict appearance of

membrane blebbing and formation of some fragmented nuclei.

At 48 h of the treatment, c cells show chromatin condensation

and cell membrane abnormality. At 72 h of the treatment, d cells

are seen with severe membrane blebbing with some fragmented

nuclei while at 96 h of treatmente formation of fragile membrane,

membrane vacuoles, and presence of apoptotic bodies can be

noticed. Each experiment was performed in triplicate and gener-

ated similar morphological features. Arrow indicates fragmented

nuclei whereas asteriskshowed apoptotic cells

Fig. 4 Graph showing the percentage of apoptotic cells in vitro

treated with different doses of cantharidin and cisplatin for 12 h.

The result is based on the AO/EtBr staining method, apoptotic

cells appears red in color whereas viable cells were green.

Thousand cells were analyzed and percentages of apoptotic cells

were counted. Results are expressed as mean SD. ANOVA,

n06, as compared to respective cisplatin treatment. *P0.05

and #P0.001

Cell Biol Toxicol


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tumor cells were rounded in shape without any apoptotic

morphology with normal round nucleus (Fig. 5a). Can-

tharidin treatment (0.5 mg/kg/day) of mice for 24 h

(Fig. 5b) showed the appearance of constricted nucleus

with condensation of chromatin in EAC cells, appear-

ance of cytoplasmic vacuoles were also observed. Mem-

brane disorganization and severe fragmented nucleus

was observed after 48 h of treatment (Fig. 5c); while at

72 h of treatment (Fig. 5d and e), there was a reduction in

cell volume showing cell shrinkage, compaction of the

nuclear chromatin, fragmentation of nuclei, condensa-tion of the cytoplasm, and appearance of the apoptotic

bodies. At the same time, large numbers of cytoplasmic

vacuoles were also observed (Fig. 5d); magnified view

of the cells indicates abnormal swelling of mitochondria

with loss of cristae (Fig. 5e). At 96 h of treatment

(Fig. 5f), the appearance of cytoplasmic as well as mem-

brane vacuoles and complete loss of cellular framework

with gradual disintegration of plasma membrane leading

to lysis of the tumor cells was visible. Moreover, after

96 h of treatment, appearance of apoptotic bodies and

fragmented nuclei were scattered outside the cells which

indicated severe cells damage by apoptosis (Fig. 5f).

Kidney histopathology

Various histopathological features of kidney from dif-

ferent groups are presented in Fig. 6 and mentioned in

Table 3. Kidney of normal mice showed the normal

structures of the renal cortex, which comprised renal

corpuscles, glomerulus, proximal, and distal convolutedtubules. Blood vessels congestion and tubular cast were

absent (Fig. 6a and d). In the cisplatin-treated mice,

which served as positive control, kidney showed im-

mense histological damages as evidenced by the

glomerular and tubular congestion with abnormal Bow-

mans capsule, blood vessel congestion, epithelial cell

desquamation, and presence of tubular cast with few

inflammatory cells (Fig. 6b). Magnified view of glomer-

ulus depicts loss of capsular wall with abnormally

Fig. 5 Ultrastructural features of Ehrlich ascites carcinoma cells.

Tumor-bearing control (a), showing a more or less rounded shape,

normal nucleus with microvilli like processes over the cells sur-

face. Cantharidin treatment (0.5 mg/kg body weight) of mice for

24 h (b) shows the appearance of nucleus abnormality with con-

densation of chromatin, appearance of cytoplasmic vacuoles. At

48 h of treatment (c), severe fragmented nuclei with disorganized

cell membrane were noted. At 72 h of the treatment (d), formation

of cytoplasmic vacuoles, disruption in the nuclear membrane and

disintegration in the cell surface membrane is prominent; arrow

indicates the apoptotic budding of the cells. Magnified view of the

cells indicates (arrow) abnormal swelling of mitochondria with

loss of cristae (e). At 96 h of treatment, major loss of cellular

framework with both cytoplasmic (arrow) and membrane

vacuoles as well as loss of nuclear membrane leading to lysis of

cancer cells may be noted

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dispersed nucleus (Fig. 6e). In cantharidin-treated mice,the kidney tubular epithelia were exfoliated from their

underlying basement membrane and their lining cells

exhibited cytoplasmic vacuolation and pyknotic nuclei

(Fig. 6c). Some glomeruli seemed to have lost their

attachments and mesangial stromas were atrophied with

dilatation in the subcapsular space (Fig. 6f). Thus, it is

evident that cantharidin treatment caused some nephro-

toxicity and cellular damage on the host but it was lower

as compared to cisplatin.

LDH isozymes profile

The analysis of LDH isozymes patterns revealed the

presence of all the five isozymes forms (i.e., LDH-1,

LDH-2, LDH-3, LDH-4, and LDH-5) in the kidney

(Fig. 7ac). After cantharidin treatment, the expression

profile as shown by band intensity (Fig. 8) of all the

isozymes decreased (Fig. 7b) significantly as compared

to control. Cisplatin treatment caused more decrease in

the isozymes (Fig. 8) intensity as compared to that of

Fig. 6 Photomicrographs of LS of kidney of different groups. a

The histological studies of normal group showed normal glo-

merular (big arrow) and tubular ( small arrow) arrangements

with normal Bowmens capsule; b cisplatin-treated group show-

ing congested vein, damaged tubule, degenerate glomeruli with

leucocyte infiltration shown in circle and dilatation of subcap-

sular space; c cantharidin-treated group showing vacuolated

cells with pyknotic nuclei, abnormal glomeruli with subcapsular

space and leucocyte infiltration shown in circle; d magnified

view of normal group glomerulus showing normal arrangement and

compact capsular wall surrounded by renal tubules; e cisplatin-

treated glomerulus showing loss of capsular wall (big arrow) with

abnormal fragmented dispersed nucleus ( small arrow);

f cantharidin-treated glomerulus showing dilatation of subcapsular

space (big arrows) and formation of large vacuoles inside the

glomerulus (small arrows)

Cell Biol Toxicol


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cantharidin treatment (Fig. 7c). The decrease in iso-

zymes after treatments in both cisplatin and cantharidin

groups may suggest the leakage of LDH from kidneyand damage to tissue.

Discussion

Our field survey with the indigenous people of Karbi

Anglong and North Cachar Hills districts of Assam,

India revealed that the people of this region frequently

use blister beetles, M. cichorii against cancer suspected

cases. It has been reported earlier that the methanol

extract of these beetles has antitumor potential (Prasadet al. 2010b). In an attempt to understand further on the

mechanism(s) of the antitumor activity, the isolation and

characterization of bioactive compound from these

beetles was undertaken along with the evaluation of

antitumor activity of isolated compound against EAC.

Other studies to isolate cantharidin from beetleshave used different solvent system like 50% aque-

ous ethanol/methanol, chloroform, n-butanol, etc. In

the present studies, we used absolute methanol as it

is rapidly evaporated while drying the extract using

rotary evaporator. Before extraction in methanol,

the beetle powder was washed three times with

petroleum ether to remove fats and pigment which

existed in extract as it may cause hindrance in the

column run.

Table 3 Histological features from longitudinal section of kidneys of normal and different treated groups of mice

Histological features Normal

(vehicle alone)

Cisplatin treated

(8 mg/kg body weightt/day)

Cantharidin treated

(0.5 mg/kg body weight/day)

Tubular congestion +++ ++++

Tubular cast ++ ++

Epithelial disquamation ++ +++Glomerular congestion ++++ ++

Blood vessel congestion ++++ ++++

Hyperaemia of medullary part ++ ++

Inflammatory cells ++ ++++

Necrosis ++++ +++

(++++) very high, (+++) high, (++) medium, (+) low, () negative

Fig. 7 Photograph showing lactate dyhydrogenase (LDH) iso-

zymes patterns in kidney in various treatment conditions in a

slab gel. Each lane is about 0.8 cm in width. a LDH isozymes

pattern of normal control; b LDH isozymes of cantharidin

treated; c represent cisplatin treatment for 14 days. In case of

normal a, the band intensity of all isozymes were high while it

decreased significantly in treatment groups (b and c) as indicated

by band intensity shown in Fig. 8

Fig. 8 Graph showing the net band intensity of different LDH

isozymes of kidney in normal, cisplatin- and cantharidin-treated

mice. The band intensity was calculated using transilluminator

bioview UXT20M8E Gel logic 100 imaging system. The net

band intensity is mean of three different gels. As compared to

the normal, band intensity of all the five isozymes decreased

significantly. Results are expressed as meanSD. ANOVA, as

compared to normal, n03,*P0.001; #P0.05

Cell Biol Toxicol


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The findings after the simulation of different spec-

troscopic data (Fig. 1) of the isolated compound from

the present studies showed that the major bioactive

compound from M. cichorii, is cantharidin which

exhibited potent antitumor activity against EAC. The

details of the characteristic features of the identified

compound, cantharidin is given in Table 1.Cantharidin (C10H12O4) with molecular weight of

196.1 is a monoterpene anhydride having the chemical

name as 2-endo, 3-endo-dimethyl-7-oxabicyclo

(2.2.1) heptanes-2-exo, 3-exo-dicarboxylic anhydride,

most abundantly found in blister beetles (Eldridge and

Casida 1995; Wang et al. 2000). Cantharidin is

absorbed by the lipid layers of cell membranes (Moed

et al. 2001). It was observed that cantharidin-treated

EAC-bearing mice showed about 82% increase in life

span (Table 2). This antitumor effect is in conformity

with the earlier reports showing the presence of can-tharidin as the major component ofM. cichorii having

anticancer potentials. Cantharidin, a vesicant produced

by beetles in the order Coleoptera, has a long history

in both folk and traditional medicine. Cantharidin has

been reported to produce cytotoxic effects in a number

of human cancer cell lines and primary cancer cells

(Huan et al. 2006; Nikbakhtzadeh and Ebrahimi 2007;

Rauh et al. 2007). The use of cantharidin and its analogs

in cancer therapy has been widely suggested (Cirrito and

Bergstein 2008). The first documented use of canthari-

din to treat cancer is by the physician Yang Shi-Yingdating back to 1264 (Wang 1989). Cantharidin has been

shown to be active in cervical, tongue, gingival, mucoe-

pidermoid carcinoma, adenocystic carcinoma, neuro-

blastoma, bone, ovarian, and colon cancer cell lines

among others (Wu et al. 1992; McCluskey et al. 2000;

Sakoff et al. 2002). Effect of cantharidins against hepa-

tocellular and colorectal tumors (Wang et al. 2000; To et

al. 2005; Chen et al. 2005) and leukemic stem cells

(Dorn et al. 2009) has been well documented.

Modifications of cantharidins skeleton permitted the

development of a new series of analogs (McClusky et al.2003; Hill et al. 2007; Liu and Zhiwei 2009). Analogs

possessing good protein phosphatases 1 (PP1) and 2A

(PP2A) inhibition exhibited good anticancer activity

(McClusky et al. 2003). PP1 and PP2A are serine/thre-

onine protein phosphatases which are inhibited by can-

tharidin (Honkanen 1993; Efferth 2005; Li and Casida

1992). Protein serine/threonine phosphatases have re-

cently been used as viable therapeutic targets in the

development of drugs (McConnell and Wadzinski

2009). Other natural product extracts have proven to

be a rich source of small molecules that potently

inhibit the activity of family ser/thr protein phos-

phatases. Some of these inhibitors include, okadaic

acid (produced by marine dionoflagelates, Prorocen-

trum sp. and Dinophysis sp.), calyculin A, dragma-

cidins (isolated from marine sponges), microcystins,nodularins (isolated from cyanobacteria, Microcystis

sp. and Nodularia sp.), tautomycin, tautomycetin,

cytostatins, phospholine, leustroducsins, phoslacto-

mycins, and fostriecin (isolated from soil bacteria,

Streptomyces sp.). Fostriecin and cantharidin pos-

sess antitumor activity, but okadaic acid and micro-

cystin LR have been touted to act as tumor-promoting

agents. Microcystin, a nonselective inhibitor primarily

affects the liver, causing minor to widespread damage,

depending on the amount of toxin absorbed (Swingle et

al. 2007).Cantharidin has also been reported to cause delays

in cell cycle progression following DNA replication

with no apparent effect on G(1)-S or S-G(2) phase

progression (Bonness et al. 2006). However, canthar-

idin can rapidly arrest growth when added during G(2)

or early M phase (Dongwu and Zhiwei 2009). Refer-

ence drug used in present studies was cisplatin which

has been established to be one of the most effective

cancer chemotherapeutic agents. It has been well

documented that cellular DNA could be the primary

target of cisplatins anticancer activity. Cisplatin iswater-soluble square planar coordination complex

containing a central platinum atom surrounded by

two-chloride atoms and two ammonia moieties.

Cisplatin is an alkylating drug and its anticancer ac-

tivity has been attributed mainly to its ability to bind

with cellular DNA involving intrastrand and inter-

strand cross-links (Fuertes et al. 2003). Cisplatin used

here as a reference drug showed ILS value of about

87% which was quite close to the ILS, 82% noted for

cantharidin treatment (Table 2). It may be of impor-

tance to mention that almost similar anticancer activityobserved with cantharidin is at much lower concentra-

tion as compared to reference drug, cisplatin. The

cytotoxic effects of cantharidin on EAC cells in in

vitro were evident at 1 h of continuous exposure. In

vitro cytotoxicity assay with the MTT test revealed an

IC50 at 25.8 g/ml while for cisplatin under the same

conditions, it was 32 g/ml (Fig. 2). This may suggest

that as compared to cisplatin, cantharidin is able to

cause heightened injury to cells and this may be due to

Cell Biol Toxicol


12/15

a better diffusion of cantharidin through the cell mem-

branes, due to its nonpolar nature and low molecular

size.

Uncontrolled proliferation and a defect in apoptosis

constitute crucial elements in the development and

progression of malignant tumors (Bryan et al. 2011;

Finkel et al. 2007). Among many other biologicalresponse modifiers known to influence these mecha-

nisms, the efficacy of drugs in the treatment of various

malignant entities is currently matter of discussion

(Bao-ying et al. 2011; Zhang et al. 2010). Apoptosis

is formally defined by morphological criteria and this

remains an important means of characterizing an apo-

ptotic cell (Kerr et al. 1972). The assay based on TEM

and AO/EtBr fluorescence staining is a good reliable

analysis for the authentication of apoptotic features

(Zakeri et al. 1995; Mattes 2007) compared to other

methods (Leite et al. 1999). Cantharidin treatmentcaused changes in cellular morphology, including

chromatin condensation, membrane blebbing, frag-

mented nuclei, large size cytoplasmic, and membrane

vacuoles with complete loss of membrane integrity

(Fig. 3). The ultrastructure of cantharidin-treated

EAC cells depicted typical apoptotic morphology with

chromatin condensation, fragmented nucleus into dis-

crete masses, cells shrinkage, etc. (Fig. 5) and mem-

brane blebbing as supported by fluorescence study

(Fig. 3). Finally, the whole cell buds, producing apo-

ptotic bodies, vary in size and structure. The apopticfeatures were seen in more than 70% of cells. Thus, it

may obviously be suggested that cantharidin treatment

could induce apoptosis in EAC cells.

The effects of cantharidin and cantharidin derivates

on tumor cells have been illustrated which also indi-

cated that cantharidin induces apoptosis in many types

of tumor cells (Liu and Zhiwei 2009). It has been

reported that cantharidin induces caspase-3, -8, and -

9 activities in myeloma cells (Sagawa et al. 2008) and

it inhibits the activity of serine/threonine protein phos-

phatase 4 (PP4; Cohen et al. 2005). During apoptosis,many functional molecules may undergo post-

translational modification, including phosphorylation,

dephosphorylation, and caspase cleavage. Some

apoptosis-regulating genes also undergo alternative

splicing, generating splice variants that antagonize

normal transcripts on apoptosis (Hoof and Goris

2003). It is also found that PP2A acts in the apoptotic

signal transduction pathway not only upstream but

also downstream of the effector caspases. PP2A

activates pro-apoptotic and inhibits anti-apoptotic pro-

teins of the Bcl-2 family; hence, PP2A is involved in

the regulation as well as the cellular response of apo-

ptosis. Probably, various PP2A holoenzymes with dis-

tinct regulatory subunits altering the PP2A substrate

specificity are implicated at different levels of the

apoptotic signal transduction pathway (Hoof andGoris 2003).

It has been found that the mitogen-activated protein

kinase (MAPK) family, the MAPK ERK kinase and

ERK become active after cantharidic acid stimulation,

and result in a significant increase in caspase-3-

mediated apoptosis of tumor cells (Schweyer et al.

2007). The cancer cells that are treated with canthari-

din may also undergo death by autophagy. For exam-

ple, breast carcinoma cells that are treated with the

estrogen-receptor antagonist tamoxifen accumulate

autophagic vacuoles shortly before dying. Extensiveautophagic degradation of the Golgi apparatus, poly-

ribosomes, and endoplasmic reticulum were seen in

high magnification (Fig. 5e and f). Morphologically,

the dead cells lack the features of cells that have

undergone apoptosis. Unusually large size of putative

autophagic vacuoles may also provide a clue to an

apoptotic origin (Abedin et al. 2007).

The full use of cisplatin for the management of

cancer is usually limited by the development of neph-

rotoxicity (Borch 1987). Histological changes in kid-

ney after cantharidin treatment of mice revealedtubular necrosis, atrophy of glomerulus, and marked

dilation of proximal convoluted tubules with slogging

of almost entire epithelium due to desquamation of

tubular epithelium which indicate injury to kidney.

There was an increase/infiltration of inflammatory

cells in the kidney after treatment (Table 3) which

may also indirectly suggest the renal irregularity/

toxicity. The simultaneous decrease in LDH isozymes

from kidney and histological abnormality is a fair

indication of altered membrane permeability of cells

in kidney. A correlation between tissue cytotoxicityand LDH release has been demonstrated and used as a

parameter of tissue damage by many workers (Takema

et al. 1991; Hasan et al. 2005). Slight damage to the

plasma membrane will easily lead to leakage of LDH

from the cell to the extracellular environment (Akanji

et al. 2008). However, the decrease in kidney LDH

activity as shown by band intensity in present study

(Fig. 8) may be due to labialized plasma membrane

(Akanji et al. 1993). At the same time, the possibility

Cell Biol Toxicol


13/15

of decreased synthesis and/or increase leakage from

the cells due to cell membrane injury may also exist.

The release of LDH is higher after cisplatin treatment

as compared to that of cantharidin suggesting more

nephrotoxic effects induced by cisplatin. It has been

reported that LDH-1 and LDH-2 isoenzymes can be

released by cellular injury to cardiac muscle or kidney(Kopperschlager and Kirchberger 1996; Akanji and

Yakubu 2000). In the present studies, it is also found

that the band intensity of LDH-1, LDH-2, and LDH-5

decreases significantly after treatment as compared to

control, supporting the damage to kidney.

In conclusion, the result of the present studies showed

that cantharidin-mediated anticancer activity against

EAC may involve apoptosis. Cantharidin treatment

caused plasma membrane disintegration and the appear-

ance of membrane vacuoles and blebbing on the tumor

cells which may lead to cell death. It also exerts somekidney damage in the host. However, the detailed mo-

lecular mechanism(s) involved in the antitumor activity

of cantharidin against EAC needs to be elucidated.

Acknowledgments We acknowledge the University Grants

Commission, New Delhi (India) for providing Research fellowship

in science for meritorious students to A.K. Verma. The electron

microscope facility was provided by Sophisticated Analytical

Instrument Facility (SAIF), North-Eastern Hill University, Shillong.

The spectroscopic and NMR facility was provided by North-East

Institute of Science and Technology, Jorhat, India. We are also

thankful to all traditional healers of Karbi Anglong and North

Cachar Hill district of Assam (India) who helped and shared the

required information during field survey and beetles collection.

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